A recently described proto-oncogene family, the ski family, may have important functions in early embryonic development, which will be investigated in 1OT1/2 cells, ES cells, and transgenic mice. The proposed studies are directed towards understanding the role of the ski proto-oncogene family in skeletal and cardiac muscle stem cell determination and differentiation in early development. Members of the ski family of genes, ski and sno, are expressed during skeletal muscle development, in determined myoblasts and in differentiated myofibers, and at lower levels in adult muscle. The chicken v-ski oncogene has been shown to transform quail embryo cells to tumorigenicity, and can also convert these non-myogenic cells to the myogenic (muscle) pathway. Several regulatory genes have been recently described that convert fibroblasts to the myogenic pathway; MyoD1, myogenin, and myf5, all of which share a basic/myc homology region crucial for the conversion function. Ski has myogenic conversion activity like the other myogenic regulatory genes, but lacks the basic/myc domain and thus represents a different class of muscle regulatory genes. Ski, unlike the MyoD1 family of myogenic regulatory genes, is expressed during cardiac muscle development and in adult cardiac cells, suggesting a potential role for ski in the formation of cardiac muscle. Identification of the different genes that regulate the commitment of stem cells to a specific lineage, and analysis of the interactions between these proteins in the skeletal and cardiac muscle lineages, may reveal important regulatory mechanisms in normal early embryonic development and provide insight into some developmental abnormalities. The specific goals of the project are to 1) characterize two new human ski homologues by nucleotide sequencing and isolate the ski gene family counterparts from mice; 2) analyze the developmental expression patterns of the ski family genes and their alternatively spliced isoforms during development, using Northern blots, S1 mapping, PCR and in situ hybridization; 3) analyze the ability of ski family and alternatively spliced isoforms and in vitro mutagenized constructs to convert cells (10T1/2 or ES) to the cardiac or skeletal myogenic pathways; 4) characterize potential interactions between ski and MyoD1 family proteins, oncogenes and growth factors, including the ability of each to induce the expression of others and the ability of ski to autoregulate itself; 5) test the DNA binding activity and specificity of ski in comparison with the other muscle regulatory proteins; 6) and to express ski family genes in transgenic mice under the control of various promoters, and mutagenize the endogenous ski genes in mice using homologous recombination and analyze the consequences.